Serine proteases are essential for a variety of biological processes, including food digestion, hormone processing, blood coagulation, complement activation, wound healing, and embryonic development (Davie, E. W. et al. (1991) Biochemistry 30:10463–10370; Kraut, J. (1977) Annu. Rev. Biochem. 46:331–358; Neurath, H. (1986) J. Cell Biochem. 32:35–49; and Stroud, R. M. (1974) Sci. Am. 231–74–88). Most trypsin-like serine proteases are secreted proteins, but several type II transmembrane proteins containing a trypsin-like protease domain at their extracellular C-terminus have been described (Hooper, J. D. et al. (2001) J. Biol. Chem. 276:857–860).
Corin is a member of the type II transmembrane serine protease class of the trypsin superfamily composed of multiple structurally distinct domains (Yan, W. et at. (1999)J. Biol. Chem. 274: 14926–14935). Mature corin is a polypeptide of 1042 amino acids (SEQ ID NO:2) consisting of a cytoplasmic tail at its N-terminus, followed by a transmembrane domain (TM, amino acids 46 to 66), a stem region composed of two frizzled-like cysteine-rich domains (CRD, amino acids 134 to 259 and 450 to 573), eight low density lipoprotein receptor repeats (LDLR, amino acids 268 to 415 and 579 to 690), a macrophage scavenger receptor-like domain (SRCR, amino acids 713 to 800, and a serine protease catalytic domain at its C-terminus (CAT, amino acids 802 to 1042) (Yan, W. et at. (1999) supra). The overall topology of corin is similar to that of other type II transmembrane serine proteases, such as hepsin (Leytus, S. P. et at. (1988) Biochemistry 27: 1067–1074) and enterokinase (Kitamoto, Y. et at. (1994) Proc. Natl. Acad. Sci. USA 91:7588–7592); however, the combination of domain structures in corin appears to be unique.
Corin contains two LDLR motifs, one with 5 repeats and the other with 3 repeats, and a SRCR motif. The LDLR and SRCR motifs are also found in other type II transmembrane serine proteases, for example, enterokinase, matriptase and TMPRSS2-4 (Hooper, J. D. et al. (2001) supra). Corin, the only trypsin-like serine protease to date that contains frizzled-like cysteine-rich domains, has two such CRD repeats, each about 120 amino acids in length containing 10 conserved cysteine residues. The functions of the CRD, LDLR, and SRCR motifs in corin remain to be defined.
The protease catalytic domain of corin is homologous to those of trypsin-like serine proteases. Amino acid sequence identities between corin and prokallikrein (Chung, D. W. et al. (1986) Biochemistry 25:2410–2417), factor XI (Fujikawa, K. et al. (1986) Biochemistry 25:2417–2424) and hepsin (Leytus, S. P. et al. (1988) supra) are 38–40%. The essential features of trypsin-like serine proteases are conserved, including the residues of the catalytic triad His843, Asp892, and Ser985 of SEQ ID NO:2, and the residues forming the substrate specificity pocket Asp979, Gly1007, and Gly1008 of SEQ ID NO:2 (Yan, W. et al. (1999) supra). The substrate specificity of corin is predicted to favor basic residues in the P1 position. The sequence Arg801-Ile802-Leu803-Gly804-Gly805 of SEQ ID NO:2, or RILGG (SEQ ID NO:42), represents a conserved activation cleavage site, indicating that proteolytic cleavage of the peptide bond between Arg801 and Ile802 is required to generate a catalytically active corin enzyme. The presence of residues Cys790 and Cys912 indicates that the catalytic domain of corin remains attached to the cell surface by a disulfide bond after the activation cleavage at Arg801. The enzyme responsible for the activation of corin under physiological conditions in vivo is not known.
Native corin appears as a major band of ˜130 kDa in SDS-PAGE and Western blot analysis (Hooper, J. D. et al. (2001) supra), consistent with the calculated mass of 116 kDa for recombinant human corin containing 19 potential N-linked glycosylation sites in its extracellular domains (Yan, W. et al. (1999) supra).
Atrial natriuretic peptide (ANP) and brain-type natriuretic peptide (BNP) are circulating peptide hormones produced primarily by cardiomyocytes in the atrium and ventricle, respectively (Levin, E. R. et al. (1998) N. Engl. J. Med. 339:321–328; Wilkins, M. R. et al. (1997) Lancet 349:1307–1310). In target organs such as kidney and peripheral vessels, these peptide hormones bind to their receptors and stimulate the intrinsic guanylate cyclase activity of the receptor, leading to production of intracellular cGMP. The biological effects of ANP and BNP are to promote salt excretion, reduce blood volume, and decrease vascular resistance, thereby reducing blood pressure (de Bold, A. J. et al. (1981) Life Sci. 28:89–94; Inagami, T. (1989) J. Biol. Chem. 264:3043–3046; Brenner, B. M. et al. (1990) Physiol. Rev. 70:665–699; Rosenzweig, A. and Seidman, C. E. (1991) Annu. Rev. Biochem. 60:229–255; Wilkins, M. R. et al. (1997) supra; Stein, B. C. and Levin, R. I. (1998) Am. Heart J. 135:914–923; Liang, F. et al. (1997) J. Biol. Chem. 272:28050–28056). BNP also has a local antifibrotic effect in the heart (Tamura, N. et al. (2000) Proc. Natl. Acad. Sci. USA 97:4239–4244). The biological importance of the ANP-mediated pathway in maintaining normal blood pressure has been demonstrated in a number of studies. In knockout mice, for example, deficiency of either ANP or its receptor leads to spontaneous hypertension (John, S. W. et al. (1995) Science 267:679–681; John, S. W. et al. (1996) Am. J. Physiol. 271:R109-R114; Lopez, M. J. et al. (1995) Nature 378:65–68).
ANP and BNP are synthesized as pre-pro-peptides in cardiomyocytes, and proteolytic cleavage is required to convert the precursors to biologically active peptide hormones (Shields, P. P. and Glembotski, C. C. (1988) J. Biol. Chem. 263:8091–8098; Ito, T. et al. (1988) Proc. Natl. Acad. Sci. USA 85:8365–8369). In response to volume overload or a hypertrophic signal, the heart increases its release of these hormones, which in turn reduce blood volume and lower blood pressure. The increased release of ANP and BNP has been attributed to the increased synthesis of pro-ANP and pro-BNP (McBride, K. and Nemer, M. (2001) Can. J. Physiol. Pharmacol. 79:673–681).
In cardiac myocytes, ANP is synthesized as a 126 amino acid pre-pro-peptide (Schwartz, D. et al. (1985) Science 229:397–400; Thibault, G. et al. (1987) Biochem. J. 241:265–272). After the signal peptide is removed, pro-ANP is stored in the dense granules of the cell. Upon secretion from the dense granules, pro-ANP is activated on the surface of cardiac myocytes by proteolytic cleavage at Arg98, generating an N-terminal pro-peptide and a biologically active, mature 26 amino acid C-terminal peptide (Shields, P. P. and Glembotski, C. C. (1988) supra; Ito, T. et al. (1988) supra). BNP is also synthesized as a pre-pro-peptide and cleavage is required to produce the mature, active peptide. The activation cleavage sequence in pro-BNP is similar to that of pro-ANP, with proteolytic cleavage occurring at Arg76. Several studies indicate that a high-molecular-weight trypsin-like enzyme associated with the membrane of cardiac myocytes is responsible for the activation cleavage of pro-ANP (Seidah, N. G. et al. (1986) Biosci. Rep. 6:835–844; Imada, T. et al. (1988) J. Biol. Chem. 263:9515–9519; Sei, C. A. et al. (1992) Mol. Endocrinol. 6:309–319).
Corin cDNA was first identified by searching genomic databases for expressed sequence tags (ESTs) that share homology with trypsin-like proteases, and was subsequently cloned from a human heart library (Yan, W. et al. (1999) supra). Northern blot and in situ hybridization analyses show that corin mRNA is highly expressed in tissues where ANP and BNP peptides are produced, predominantly in the atrium and ventricle of the heart (Yan, W. et al. (1999) supra). In functional studies (Yan, W. et al. (2000) Proc. Natl. Acad. Sci. USA 97:8525–8529; Wu, F. et al. (2002) J. Biol. Chem. 277:16900–16905), recombinant corin converts pro-ANP into biologically active ANP in a highly sequence-specific manner, indicating that corin is the pro-ANP convertase. In addition, recombinant corin processes pro-BNP to BNP (Yan, W. et al. (2000) supra).
Congestive heart failure (CHF) is a life-threatening disease, afflicting approximately 4.8 million Americans. Each year, approximately 550,000 new cases are diagnosed in the United States. Symptomatic decompensation is the most common reason for the hospitalization of patients with CHF due to left ventricular systolic dysfunction. Most common symptoms include dyspnea and fatigue resulting mainly from pulmonary venous congestion and reduced cardiac output. Traditionally, patients with decompensated CHF are treated with diuretics, inotropic agents, vasodilators and β blockers (Braunwald, E. et al. (1997) in Heart Disease: A Textbook of Cardiovascular Medicine, Braunwald, E., ed., W. B. Saunders, Philadelphia, pp.445–470). The therapeutic goal is to increase sodium and fluid excretion, decrease cardiac filling pressures, reduce peripheral vascular resistance, and increase cardiac output. The various drugs used to treat CHF usually alleviate symptoms, although each therapy has inherent limitations. For example, inotropic drugs increase cardiac contractility and oxygen consumption of the failing heart, but also increase the incidence of cardiac arrhythmias and may increase mortality. Repeated and prolonged use of nitroglycerin, a vasodilator originally manufactured by Alfred Nobel to produce dynamite over 130 years ago, results in nitrate tolerance with the loss of clinical efficacy over time, leaving patients vulnerable to new ischemic attacks.
Despite advances in the understanding of the pathophysiology of CHF, effective treatments for advanced disease are limited and the morbidity and mortality remains high. Current approaches to chronic treatment of CHF target the renin-angiotension system (angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers), the sympathetic nervous system (β-blockers), endothelin-1 (ET-1) (ET-1 receptor antagonists) and the natriuretic peptide system (ANP, BNP, and neutral endopeptidase (NEP) inhibitors) (McMurray, J. and Pfeffer, M. A. (2000a) Circulation 105:2099–2106; McMurray, J. and Pfeffer, M. A. (2000b) Circulation 105:2223–2228; Corti, R. et al. (2001) Circulation 104:1856–1862). Clinically, high plasma concentrations of ANP and BNP are found in patients with CHF. The levels of these natriuretic peptides are often correlated with the extent of ventricular dysfunction and development of cardiac arrhythmias (Burnett, J. C., Jr. et al. (1986) Science 231:1145–1147; Gottleib, S. S. et al. (1989) J. Am. Coll. Cardiol. 13:1534–1539). BNP is currently used as a diagnostic and prognostic marker in CHF (Gottleib, S. S. et al. (1989) supra; Maisel, A. S. et al. (2002) N. Engl. J. Med. 347:161–167). Natrecor, a recombinant form of human BNP, has been approved in the U.S. as a short-term, in hospital treatment for decompensated CHF. Infusion of BNP has been shown to be more effective than nitroglycerin in improving hemodynamics and cardiac and renal function in patients with CHF (Colucci, W. S. et al. (2001) supra). ANP has also been used in Japan to treat patients with CHF and renal failure (Hayashi, M. et al. (2001) supra; Mizuno, O. et al. (2001) J. Am. Cardiol. 88:863–866; Allgren, R. L. et al. (1997) N. Engl. J. Med. 336:828–834). The results with ANP and BNP demonstrate that natriuretic peptide-based therapies are effective in relieving symptoms and improving hemodynamics and cardiac function in patients with severe CHF.
The discovery of corin provides an opportunity to use recombinant corin as a biological agent to increase the production of both ANP and BNP in vivo. Corin-based therapy may be more effective than either ANP or BNP alone in the treatment of decompensated CHF. In addition, corin may offer pharmacokinetic advantages over ANP or BNP, which must be administered by continuous, intravenous infusion. The instant invention is a novel soluble form of corin as a biological therapy for decompensated CHF.